Method of measuring total homocysteine

ABSTRACT

The present invention provides a method and a kit for detecting or quantitatively determining homocysteine rapidly and simply and with high sensitivity by oxidizing the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof in the presence of an SH reagent to produce hydrogen peroxide and determining the produced hydrogen peroxide by color development using an oxidative color-developing agent. By using the method and kit of the present invention, homocysteine in biological samples, in particular, in body fluids such as blood and urine can be detected and quantitatively determined rapidly and simply and with high sensitivity.

TECHNICAL FIELD

[0001] The present invention relates to a method for determining totalhomocysteine.

BACKGROUND ART

[0002] Homocysteine, which is one of the metabolic intermediates inmethionine metabolism, is reported to have vascular endothelialcytotoxicity and to be one of risk factors for arteriosclerotic diseasesindependent from the other risk factors. It also has been evident thatin addition to serious hyperhomocysteinemia (homocystinuria) caused bydeficiency of homocysteine metabolic enzymes, moderatehyperhomocysteinemia is caused by a decrease in the metabolic enzymeactivity due to abnormality of genes, renal insufficiency, aging,smoking, lack of exercise or the like (Jacobsen, Clin. Chem. 44:8(B),1998). Furthermore, hyperhomocysteinemia is also reported to be improvedby taking vitamin B6, folic acid or the like (JAMA 270: 693, 1993).Therefore, not only for neonatal mass screening, but also for preventionof adult arteriosclerotic diseases or detection of vitamin deficiencydiseases, a simple method for treating a large number of specimens is indemand.

[0003] Most of homocysteine in blood (99%) is present in the form ofoxidized disulfide compounds (such as complex with protein, homocystine,cysteine-homocysteine) (Jacobsen, Clin. Chem. 44:8(B), 1998). “Totalhomocysteine” refers to the total amount of oxidized and reducedhomocysteines, and in general, it is necessary to convert homocysteinein a sample to reduced homocysteine by a reducing agent in order todetermine the total homocysteine.

[0004] High-performance liquid chromatography (HPLC) and immunoassay areusually employed to determine homocysteine. However, the HPLCapparatuses used in high-performance liquid chromatography are notcommonly used in the clinical test, and it takes time, labor and cost tooperate the apparatuses. In the immunoassay, although apparatuses areautomated (Shipchandler, Clin. Chem., 41, 7, 991-994, 1995),determination is performed by combining a process for convertinghomocysteine to S-adenosyl-L-homocysteine by an enzyme reaction and aprocess for detecting it by an immunoassay, so that an apparatus usedexclusively for this purpose is required.

[0005] Determination methods of homocysteine based on an immunoassay areproposed in Japanese Laid-Open Patent Publication (Tokuhyo) No. 9-512634and (Tokkai) No. 10-114797. In the method disclosed in JapaneseLaid-Open Patent Publication No. 9-512634, homocysteine is determinedimmunologically by chemically modifying the homocysteine to enhance theantigenicity, which requires a large number of processes and iscomplicated. Japanese Laid-Open Patent Publication No. 10-114797discloses a method for determining homocysteine, but this methoddirectly determines only the homocysteine bound to albumin, and does notdetermine the total amount of homocysteine. In this method, only about70% of the entire homocysteine can be determined.

[0006] On the other hand, biochemical determination methods ofhomocysteine are disclosed in Japanese Patent No. 2870704, U.S. Pat.Nos. 5,998,191 and 5,885,767. The method disclosed in Japanese PatentNo. 2870704 is characterized by allowing homocysteine in a sample thathas been treated with a reducing agent to be in contact with adenosineand S-adenosyl-L-homocysteine hydrolase and evaluating the amount ofadenosine in the residual mixture. However, in this method, an inhibitorof the S-adenosyl-L-homocysteine hydrolase is not used, and therefore itis necessary to perform determination in kinetic mode. Furthermore, thismethod has the problem that produced hydrogen peroxide cannot be led toa commonly used oxidative color-developing agent in the presence of areducing agent that is used for a reduction process, which is anessential process for determining the total homocysteine. Therefore, anautomatic analysis apparatus for general purposes cannot be used.However, these patent specifications fail to disclose any method toavoid these problems.

[0007] The methods disclosed in Japanese Laid-Open Patent Publication(Tokuhyo) No. 2000-502262, U.S. Pat. Nos. 5,998,191 and 5,885,767 arecharacterized by reacting homocysteine with homocysteine desulfurase,homocysteinase, or methionine-γ-lyase to detect the produced hydrogensulfide, ammonia, or 2-oxobutyric acid. However, these methods haveproblems such as: requiring a large number of processes; employing alead ion, which is a harmful heavy metal, for the detection of thehydrogen sulfide; and being affected by cysteine and methionine, whichare structural analogs to homocysteine and contained in a biologicalsample in a larger amount than that of homocysteine.

[0008] Thus, the conventional methods of determinig homocysteine haveproblems such as requiring a special apparatus and complicated operationand having insufficient sensitivity and specificity, so that a methodfor determining a trace concentration of homocysteine rapidly, simplyand with high sensitivity has not been established yet.

DISCLOSURE OF INVENTION

[0009] Therefore, the object of the present invention is to provide anovel method for determining homocysteine contained in a biologicalsample or the like rapidly, simply and with high sensitivity, and a kitfor use in this determination method.

[0010] The inventors of the present invention succeeded in detecting ordetermining homocysteine by (i) oxidizing the residual homocysteinecosubstrate, the produced homocysteine-converting enzyme product or anenzyme reaction product thereof in the presence of an SH reagent toproduce hydrogen peroxide and detecting or determining the producedhydrogen peroxide by color development using an oxidativecolor-developing agent or (ii) reacting the residual homocysteinecosubstrate, the produced homocysteine-converting enzyme product or anenzyme reaction product thereof with a D-amino acid converting enzyme toproduce an oxo acid and/or ammonia and detecting or determining theproduced oxo acid and/or ammonia. With the method for determininghomocysteine of the present invention, homocysteine in a biologicalsample, in particular in body fluids such as blood and urine can bedetected and determined rapidly and simply.

[0011] The present invention is directed to a method for detecting ordetermining homocysteine in a sample including:

[0012] (a) reducing the homocysteine in the sample by a thiol compound,

[0013] (b) reacting the reduced homocysteine with ahomocysteine-converting enzyme and a homocysteine cosubstrate, therebyproducing a homocysteine-converting enzyme product, and

[0014] (c) detecting or determining the residual homocysteinecosubstrate, the produced homocysteine-converting enzyme product or anenzyme reaction product thereof by: (i) oxidizing the residualhomocysteine cosubstrate, the produced homocysteine-converting enzymeproduct or an enzyme reaction product thereof in the presence of an SHreagent to produce hydrogen peroxide and detecting or determining theproduced hydrogen peroxide by color development using an oxidativecolor-developing agent or (ii) reacting the residual homocysteinecosubstrate, the produced homocysteine-converting enzyme product or anenzyme reaction product thereof with a D-amino acid converting enzyme toproduce an oxo acid and/or ammonia and detecting or determining theproduced oxo acid and/or ammonia.

[0015] In one preferable embodiment, the homocysteine-converting enzymein the step (b) is S-adenosyl-L-homocysteine hydrolase, and thehomocysteine cosubstrate in the steps (b) and (c) is adenosine.

[0016] In one preferable embodiment, the step (c) of detecting ordetermining the adenosine is a step of detecting or determining theadenosine by reacting the adenosine with adenosine deaminase, phosphoricacid, purine nucleoside phosphorylase, and xanthine oxidase to producehydrogen peroxide and detecting or determining the produced hydrogenperoxide by color development using peroxidase and an oxidativecolor-developing agent.

[0017] In a more preferable embodiment, the step (c) includes furtherreacting the adenosine with uricase.

[0018] In another more preferable embodiment, thehomocysteine-converting enzyme in the step (b) is a methyltransferaseusing homocysteine as a methyl acceptor, and the homocysteinecosubstrate in the steps (b) and (c) is a methyl donor.

[0019] In one preferable embodiment, the methyltransferase ishomocysteine methyltransferase, and the methyl donor is D-methioninemethyl sulfonium.

[0020] In a more preferable embodiment, in the step (c), thehomocysteine-converting enzyme product is D-methionine, and theD-methionine is determined by reacting it with a D-amino acid convertingenzyme.

[0021] In a more preferable embodiment, the D-amino acid convertingenzyme is D-amino acid oxidase.

[0022] In one preferable embodiment, in the step (c), the hydrogenperoxide produced by a reaction with the D-amino acid oxidase isdetected or determined by color development using peroxidase and anoxidative color-developing agent.

[0023] In one preferable embodiment, the SH reagent is a maleimidederivative.

[0024] In another preferable embodiment, in the step (c), a decrease inNAD(P)H or an increase in NAD(P) is detected or determined by reactingthe produced oxo acid and/or ammonia with dehydrogenase using NAD(P)H asa coenzyme.

[0025] In one preferable embodiment, in the step (c), the oxo acidand/or ammonia produced by a reaction with the D-amino acid oxidase isdetected or determined.

[0026] In one preferable embodiment, in the step (c), a decrease inNAD(P)H or an increase in NAD(P) is detected or determined by reactingthe produced oxo acid and/or ammonia produced by a reaction with theD-amino acid oxidase with dehydrogenase using NAD(P)H as a coenzyme.

[0027] In one preferable embodiment, the dehydrogenase is leucinedehydrogenase, and a decrease in NAD(P)H is detected or determined byreacting the produced oxo acid with the leucine dehydrogenase in thepresence of ammonia and NAD(P)H.

[0028] In one preferable embodiment, the dehydrogenase is lactatedehydrogenase, and a decrease in NAD(P)H is detected or determined byreacting the produced oxo acid with the lactate dehydrogenase in thepresence of NAD(P)H.

[0029] In one preferable embodiment, the dehydrogenase is glutamatedehydrogenase, and a decrease in NAD(P)H is detected or determined byreacting the produced ammonia with the glutamate dehydrogenase in thepresence of 2-oxoglutaric acid and NAD(P)H.

[0030] In a more preferable embodiment, the dehydrogenase is lactatedehydrogenase and glutamate dehydrogenase, and a decrease in NAD(P)H isdetected or determined by a reaction with the lactate dehydrogenase andthe glutamate dehydrogenase in the presence of 2-oxoglutaric acid andNAD(P)H.

[0031] In a more preferable embodiment, the steps (a) and (b) areperformed at the same time.

[0032] The present invention also is directed to a reagent kit forhomocysteine determination comprising a thiol compound, ahomocysteine-converting enzyme, a homocysteine cosubstrate, an SHreagent, and an oxidative color-developing agent.

[0033] In one preferable embodiment, the SH reagent is contained inanother container from one for the thiol compound, thehomocysteine-converting enzyme and the homocysteine cosubstrate.

[0034] In one preferable embodiment, the homocysteine-converting enzymeis contained in another container from one for the homocysteinecosubstrate.

[0035] In a more preferable embodiment, the homocysteine-convertingenzyme is S-adenosyl-L-homocysteine hydrolase, and the homocysteinecosubstrate is adenosine.

[0036] In an even more preferable embodiment, the kit further includesadenosine deaminase, phosphoric acid, purine nucleoside phosphorylase,xanthine oxidase, and peroxidase.

[0037] In another preferable embodiment, the adenosine deaminase iscontained in another container from one for the thiol compound, theS-adenosyl-L-homocysteine hydrolase and the adenosine.

[0038] In one preferable embodiment, the kit further includes uricase.

[0039] The present invention also is directed to a reagent kit forhomocysteine determination comprising a thiol compound, ahomocysteine-converting enzyme, a homocysteine cosubstrate, and aD-amino acid converting enzyme.

[0040] In one preferable embodiment, the kit further includes NAD(P)H,dehydrogenase using NAD(P)H as a coenzyme, and an ammonium salt or 2-oxoacid as its cosubstrate.

[0041] In a more preferable embodiment, the dehydrogenase is leucinedehydrogenase, and the cosubstrate of the enzyme is an ammonium salt.

[0042] In another more preferable embodiment, the dehydrogenase isglutamate dehydrogenase, and the cosubstrate of the enzyme is a2-oxoglutaric acid.

[0043] In one preferable embodiment, the dehydrogenase is lactatedehydrogenase.

[0044] In another preferable embodiment, the homocysteine-convertingenzyme is a methyltransferase using homocysteine as a methyl acceptor,and the homocysteine cosubstrate is a methyl donor.

[0045] In one preferable embodiment, the methyltransferase ishomocysteine methyltransferase, and the methyl donor is D-methioninemethyl sulfonium.

[0046] In one preferable embodiment, the D-amino acid converting enzymeis D-amino acid oxidase.

[0047] In one preferable embodiment, the D-amino acid oxidase iscontained in another container from one for the thiol compound and thehomocysteine methyltransferase.

[0048] In one preferable embodiment, the SH reagent is a maleimidederivative.

BRIEF DESCRIPTION OF DRAWINGS

[0049]FIG. 1 is a schematic diagram of the reaction whenS-adenosyl-L-homocysteine hydrolase and adenosine are used as ahomocysteine-converting enzyme and a homocysteine cosubstrate.

[0050]FIG. 2 is a schematic diagram of the reaction when homocysteinetransferase and D-methionine methyl sulfonium are used as ahomocysteine-converting enzyme and a homocysteine cosubstrate.

[0051]FIG. 3 is a graph showing the effects of adding an SH reagent onthe color development in a homocysteine determination system by the useof an oxidative color-developing agent.

[0052]FIG. 4 is a graph showing the dose dependence when a homocystinespecimen is used as a sample.

[0053]FIG. 5 is a graph showing the absorbance in each case where areagent with S-adenosyl-L-homocysteine hydrolase added is used and wherea reagent without it is used when determining homocysteine in a serumsample.

[0054]FIG. 6 is a graph showing the results of determining homocysteinein a serum sample.

[0055]FIG. 7 is a graph showing the effect of cysteine and methionine onthe homocysteine determination system.

[0056]FIG. 8 is a graph showing the correlation of the values obtainedby determining homocysteine in a serum sample by the method of thepresent invention (SAHase method) and the conventional HPLC method.

[0057]FIG. 9 is a graph showing the reaction time course of D-amino acidoxidase derived from porcine kidney with respect to D-methionine andD-methionine methyl sulfonium.

[0058]FIG. 10 is a graph showing the reaction time course of D-aminoacid oxidase derived from fungi with respect to D-methionine andD-methionine methyl sulfonium.

[0059]FIG. 11 is a graph showing the effects of an SH reagent on aD-methionine determination system by the use of an oxidativecolor-developing agent in the presence of a reducing agent.

[0060]FIG. 12 is a graph showing the dose dependence when a homocystinespecimen is used as a sample.

[0061]FIG. 13 is a graph showing the absorbance in each case where areagent with D-methionine methyl sulfonium added is used and where areagent without it is used when determining homocysteine in a serumsample.

[0062]FIG. 14 is a graph showing the results of determining homocysteinein a serum sample using a highly sensitive color-developing agent.

[0063]FIG. 15 is a graph showing the correlation of the values obtainedby determining homocysteine in a serum sample by the method of thepresent invention (MTase method I) and the conventional HPLC method.

[0064]FIG. 16 is a graph showing the dose dependence in a D-methioninedetermination system by the use of D-amino acid oxidase and leucinedehydrogenase.

[0065]FIG. 17 is a graph showing the dose dependence in a homocysteinedetermination system (the method of the present invention: MTase methodII) by the use of homocysteine methyltransferase, D-amino acid oxidaseand leucine dehydrogenase.

[0066]FIG. 18 is a graph showing the dose dependence in a homocysteinedetermination system (the method of the present invention: MTase methodII) by the use of homocysteine methyltransferase, D-amino acid oxidaseand glutamate dehydrogenase.

[0067]FIG. 19 is a graph showing the dose dependence in a4-methylthio-2-oxobutyric acid determination system by the use oflactate dehydrogenase.

BEST MODE FOR CARRYING OUT THE INVENTION

[0068] The method of the present invention is characterized by reactinghomocysteine in a sample that has been reduced by a thiol compound witha homocysteine-converting enzyme and a homocysteine cosubstrate, andthen determining a homocysteine-converting enzyme product or a residualhomocysteine cosubstrate in the presence of an SH reagent. Preferably,S-adenosyl-L-homocysteine hydrolase and adenosine are used as thehomocysteine-converting enzyme and the homocysteine cosubstrate, and theresidual adenosine is determined by using an oxidative color-developingagent in the presence of an SH reagent. In a more preferable embodiment,the method of the present invention includes a process of convertinghomocysteine in a test sample to a reduced homocysteine by a treatmentwith a thiol compound, and simultaneously reacting the homocysteine withS-adenosyl-L-homocysteine hydrolase and adenosine so as to produceS-adenosyl-L-homocysteine (hereinafter, referred to as a first process)and a process of determining the residual adenosine by using anoxidative color-developing agent in the presence of an SH reagent(hereinafter, referred to as a second process).

[0069] Alternatively, the method of the present invention ischaracterized by reacting homocysteine in a sample with amethyltransferase in the presence of a methyl donor and then determiningthe produced D-amino acid derivative or D-amino acid analog. Examples ofthe methyl donor include D-methionine methyl sulfonium,S-adenosyl-D-methionine, and D-ethionine methyl sulfonium. Preferably,D-methionine methyl sulfonium can be used.

[0070] The determination principles of the present invention will bedescribed with reference to FIG. 1 in the case (A) whereS-adenosyl-L-homocysteine hydrolase and adenosine are used as thehomocysteine-converting enzyme and the homocysteine cosubstrate, andwith reference to FIG. 2 in the case (B) where a methyltransferase andD-methionine methyl sulfonium are used as the homocysteine-convertingenzyme and the homocysteine cosubstrate.

[0071] (A) Determination principle when S-adenosyl-L-homocysteinehydrolase and adenosine are used (SAHase method)

[0072] In the first process of FIG. 1, the reduced homocysteine isreacted with S-adenosyl-L-homocysteine hydrolase and adenosine. In thisprocess, the equilibrium of the enzyme reaction is tend to the synthesisof S-adenosyl-L-homocysteine, and the adenosine is consumed togetherwith the homocysteine. Then, in the second process, the residualadenosine is reacted with adenosine deaminase, purine nucleosidephosphorylase, xanthine oxidase or the like to produce hydrogenperoxide, and a color is developed by peroxidase and an oxidativecolor-development agent for determination. As seen from FIG. 1, in thefirst process, the amount of the adenosine consumed is proportional tothe amount of the homocysteine. In the second process, as describedbelow in detail, an SH reagent is added as an inhibitor of theS-adenosyl-L-homocysteine hydrolase and a blocking agent of the thiolcompound, so that the determination can be performed with highsensitivity. Furthermore, in the second process, uricase is added sothat the production of hydrogen peroxide increases and thus thedetermination can be performed with even higher sensitivity.

[0073] Examples employing an SH reagent to determine homocysteine aredescribed in Japanese Laid-Open Patent Publication (Tokuhyo) No.9-512634 and U.S. Pat. No. 6,020,206. As described above, the former isa method including chemically modifying homocysteine to enhance theimmunogenicity and detecting it immunologically, and in the method an SHcompound is used as the modifier. The latter is a method includingconverting homocysteine to homocysteine thiolactone to protect the thiolgroup, removing other compounds having a thiol group, such as cysteinecontained in the sample, with an SH reagent, opening the ring of thethiolactone, and determining the homocysteine. Both of the methods havecompletely different determination principles from that of the presentinvention, and the SH reagent is used for a different purpose from thatof the present invention. Furthermore, for determination of free fattyacids, there are some examples where an SH reagent is used to prevent acosubstrate CoA from interfering with the determination by using anoxidative color-developing agent (Japanese Laid-Open Patent Publication(Tokkai) Nos. 55-64800 and 57-8797). However, the determination isperformed regarding different items, so that it cannot be predictedwhether or not the SH reagent is effective in the present invention.

[0074] Examples of the SH reagent include an oxidizing agent such as theEllman's reagent, a mercaptide forming agent such as p-mercuribenzoicacid, and an alkylating agent such as iodoacetic acid andN-ethylmaleimide, as described in Seikagakujiten (BiochemicalDictionary) (third edition, p. 182, Tokyo Kagaku Dojin, 1998).Preferably, an alkylating agent, and more preferably a maleimidecompound, and most preferably, N-ethylmaleimide can be used.

[0075] Any sample can be used as the test sample to be subjected to themethod of the present invention, as long as it is believed to containhomocysteine. The homocysteine can be present in the form of, not onlyreduced homocysteine, but also oxidized homocysteine that is bound toanother molecule by a disulfide bond such as a complex with protein, ahomocysteine dimer and a homocysteine-cysteine dimer. For example,serum, plasma, blood, urine and a dilution thereof can be used.

[0076] The thiol compound used in the method of the present invention isnot particularly limited, and includes, for example, dithiothreitol,mercaptoethanol, N-acetylcysteine, dithioerythritol, and thioglycolicacid. Any concentration can be employed as the concentration of thethiol compound, as long as it allows oxidized homocysteine to beconverted to reduced homocysteine. Preferably, the concentration is 0.1mM or more in terms of a thiol group, and more preferably 1 mM or more.

[0077] Following or at the same time as the reduction process by the useof the thiol compound, in the first process of the present invention, ahomocysteine-converting enzyme and a homocysteine cosubstrate,preferably, S-adenosyl-L-homocysteine hydrolase and adenosine arereacted with the homocysteine to produce S-adenosyl-L-homocysteine.

[0078] The amount of the residual homocysteine cosubstrate, preferablythe amount of the adenosine, depends on the amount of the homocysteineto be determined, as evident from the determination principle of themethod of the present invention. More specifically, in a reaction inwhich water or a buffer is used as a sample, that is, a reaction inwhich homocysteine is not contained, the amount of the adenosine is setto a value that allows the absorbance to change from 0.0005 to 4,preferably 0.001 to 2, even more preferably 0.005 to 1, from before theend of the first process to the end of the second process.

[0079] The S-adenosyl-L-homocysteine hydrolase is an enzyme forcatalyzing both a synthesis reaction and a hydrolysis reaction, which isthe reverse reaction of the synthesis reaction, and the equilibrium ofthe reaction lies significantly to the synthesis direction, but sincethe product can be metabolized rapidly in an organism, this enzymeserves as a hydrolysis system (Enzyme Handbook, p. 529, Asakura Syoten(1982)). This enzyme is isolated from various sources such as rabbits,lupine seeds, bovines, rats, yeasts, and bacteria. There is noparticular limitation regarding the source. Enzymes obtained by generecombination also can be used. The concentration employed is preferably0.01 U/mL to 100 U/mL, more preferably 0.1 U/mL to 20 U/mL.

[0080] It is preferable that this enzyme specimen is minimized tocontain an enzyme that can act on adenosine, such as adenosinedeaminase. However, some commercially available specimens containadenosine deaminase in a very small content. In this case, by purifyingthe enzyme, the effect of adenosine deaminase can be avoided.Alternatively, the effect of the contamination of the adenosinedeaminase can be avoided by determining the difference between thereactivity of the reagent containing the specimen of the enzyme(S-adenosyl-L-homocysteine hydrolase) and that of the reagent notcontaining the same.

[0081] Furthermore, in order to suppress the activity of thecontaminated adenosine deaminase, an inhibitor can be used. Anyinhibitor can be used, as long as it does not intensely act on theS-adenosyl-L-homocysteine hydrolase. However, in view of the enzymereaction in the second process as described below, coformycin,deoxycoformycin, 1,6-dihydro-6-hydroxymethylpurine ribotide(Biochemistry, 19:223, 5303-5309, 1980) and the like, which are believedto have particularly little effect on the initial rate of the adenosinedeaminase reaction, are preferable. When an inhibitor is used, it ispreferable to use an excessive amount of adenosine deaminase in thesecond process.

[0082] In the following second process, the homocysteine-convertingenzyme product or the residual homocysteine cosubstrate is determined.Preferably, first, the residual adenosine is converted to inosine by theaction of adenosine deaminase. Thus, the substrate for theS-adenosyl-L-homocysteine hydrolase reaction in the first processdecreases, that is, the product of the reverse reaction thereofdecreases, so that the equilibrium of the reaction is directed tohydrolysis. If the activity of the S-adenosyl-L-homocysteine hydrolaseremains in this process, the reverse reaction of the first process, thatis, the S-adenosyl-L-homocysteine hydrolysis reaction occurs at the sametime, which eventually leads to lower the sensitivity for homocysteinedetermination. Therefore, in order to prevent this reaction, it ispreferable to add an inhibitor of the S-adenosyl-L-homocysteinehydrolase. As inhibitors of this enzyme, the SH reagent (Eur. J.Biochem. 80, 517-523, 1977) and some adenosine derivatives (Methods inEnzymology 143, 377-383, 1987) are known. In the method of the presentinvention, any inhibitor can be used, as long as it does not cause alarge impediment in other reaction systems. The inhibitors can be usedin combination.

[0083] The produced inosine produces hydrogen peroxide by being broughtinto contact with phosphoric acid, purine nucleoside phosphorylase,xanthine oxidase, and, if necessary, uricase so that a usual oxidativecolor-developing agent can develop a color with peroxidase. This methodis generally employed in the field of clinical chemistry, but the colordevelopment is significantly interfered by the reductive action of thethiol compound used in the first process. Therefore, it is essential toadd an SH reagent, which is a blocking agent of the thiol compound, inthe second process. Thus, the addition of an SH reagent in the secondprocess serves to prevent the S-adenosyl-L-homocysteine hydrolase usedin the first process from catalyzing the reverse reaction (hydrolysisreaction) in the second process, and also serves to prevent the thiolcompound from interfering with the color development of the oxidativecolor-developing agent. Any concentration can be employed as theconcentration of the SH reagent in the second process, as long as it canblock the thiol group of the thiol compound used for a reductiontreatment of the sample from interfering with the quantitativedetermination by the use of the oxidative color-developing agent. Theconcentration can be preferably 0.1 mM to 100 mM. More preferably it is1 mM to 30 mM. It is preferable to use an excessively larger amount ofthe SH reagent than that of the thiol compound used, in order to exhibitthe effect of inhibiting the S-adenosyl-L-homocysteine hydrolase.

[0084] As the oxidative color-developing agent, various Trinder reagentscan be used in combination with a coupler reagent. This method may becalled the Trinder method, and is commonly used in the field of theclinical chemical analysis. Although the detailed description is omittedherein, it is preferable to use 4-aminoantipyrine as the coupler reagentand to use ADOS [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline],DAOS [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline], HDAOS[N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline], MAOS[N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline], TOOS[N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline] or the like as theTrinder reagent. Furthermore, leuco-type color-developing agent such aso-tolidine, o-dianisidine, DA-67[10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazinesodium, manufactured by Wako Pure Chemical Industries, Ltd.], and TPM-PS[N,N,N′,N′,N″,N″-hexa(3-sulfopropyl)-4,4′,4″-triaminotriphenylmethanehexasodium salt, Dojin Kagaku Kenkyusho], which do not require thecoupler reagent, can be used as well. In particular, DA-67 and TPM-PShave a mole absorption coefficient larger than that of the Trinderreagent, so that the determination can be performed with highersensitivity.

[0085] A reaction intermediate after inosine in the second process maybe contained in the sample, so that for the purpose of avoiding theeffect on the value to be obtained, hydrogen peroxide can be produced byusing purine nucleoside phosphorylase and phosphoric acid, which arecosubstrates thereof, and xanthine oxidase, and, if necessary, uricasein the first process. The produced hydrogen peroxide disappears by theaction of a reducing agent contained in the first process, but it ispreferable to further degrade the produced hydrogen peroxide into waterand oxygen by catalase or to produce a colorless complex by reacting theproduced hydrogen peroxide with one of the Trinder reagent or thecoupler reagent using peroxidase.

[0086] In the method of the present invention, it is possible todetermine homocysteine by using only a reagent containing theS-adenosyl-L-homocysteine hydrolase described above. However, in orderto further enhance the determination precision, by determining a reagentnot containing this enzyme at the same time, the effect of the adenosinecontained in the sample can be avoided. More specifically, the valueobtained by the determination with a reagent not containing theS-adenosyl-L-homocysteine hydrolase indicates the total amount (totaladenosine) of the amount of the adenosine previously contained in thereagent and the amount of the adenosine contained in the sample. On theother hand, the reagent containing the S-adenosyl-L-homocysteinehydrolase indicates the amount obtained by subtracting the amount of theadenosine consumed in the first process from the total amount of theadenosine described above. The difference between the thus obtained twovalues indicates the amount of the consumed adenosine, that is, theamount of homocysteine.

[0087] (B) Determination principle when a methyltransferase andD-methionine methyl sulfonium are used (methyltransferase method(hereinafter, referred to as MTase method))

[0088] Next, the determination principle of the case where amethyltransferase using homocysteine as a methyl acceptor andD-methionine methyl sulfonium, which is a methyl donor, are used will bedescribed with reference to FIG. 2. In other words, in this case, thehomocysteine in a sample is reacted with a methyltransferase andD-methionine methyl sulfonium, and then the produced D-methionine isdetermined.

[0089] (1) MTase Method I

[0090] Any methyltransferase can be used, as long as it reacts withD-methionine methyl sulfonium and L-homocysteine and catalyzes theproduction of D-methionine and L-methionine. Examples of themethyltransferase include homocysteine methyltransferase [EC 2.1.1.10],5-methyltetrahydrofolic acid-homocysteine S-methyltransferase [EC2.1.1.13], 5-methyltetrahydropteroyltriglutamic acid-homocysteineS-methyltransferase [EC 2.1.1.14]. Preferably, homocysteinemethyltransferase [EC 2.1.1.10] can be used. The phylogenetic name ofhomocysteine methyltransferase is S-adenosyl-L-methionine:L-homocysteine S-methyltransferase, and this enzyme producesL-methionine and S-adenosyl-L-homocysteine, using L-homocysteine, whichis a methyl acceptor, and S-adenosyl-L-methionine, which is a methyldonor, as the substrates (Enzyme handbook, Asakura Syoten, 1982).Furthermore, S. K. Shapiro has reported that this enzyme also utilizesS-methyl-L-methionine (L-methionine methyl sulfonium) orS-adenosyl-D-methionine as the methyl donor (Biochim. Biophys. Acta, 29,405-409, 1958).

[0091] This report also confirmed from the results of labelingexperiments with a radioisotope that methionine is produced by themethyl group of S-adenosylmethionine being transferred to homocysteine,and not by the bond between ribose and a sulfur atom of S-adenosylmethionine being opened. Therefore, when S-adenosyl-L-methionine is usedas the methyl donor, L-methionine and S-adenosyl-L-homocysteine isproduced. When L-methionine methyl sulfonium is used as the methyldonor, two L-methionine molecules are produced. WhenS-adenosyl-D-methionine is used as the methyl donor, L-methionine andS-adenosyl-D-homocysteine is produced. In all the cases, L-methionine isproduced, so that the amount of the homocysteine can be quantitativelydetermined by determining the L-methionine.

[0092] However, in general, L-methionine is contained in a biologicalsample in a larger amount than that of homocysteine (3 to 5 times largerin plasma), so that it is necessary to remove L-methionine previously ina manner that does not affect homocysteine and to determine specificallythe L-methionine that is produced by a homocysteine methyltransferasereaction, which is a complicated operation, and therefore this method isnot preferable. On the other hand, G. Grue-Sorensen et al. have reportedthat homocysteine methyltransferase produces D-methionine by usingD-methionine methyl sulfonium as the methyl donor (J. Chem. Soc. PerkinTrans. I 1091-7 (1984)), although the specificity is low. For thisreason, in the method of the present invention, utilizing this reaction,D-methionine, which is not substantially present in a biological sample,is produced, and then the D-methionine is determined so as toquantitatively determine homocysteine specifically.

[0093] Homocysteine methyltransferase derived from any sources can beused as the homocysteine methyltransferase for the present invention, aslong as it uses D-methionine methyl sulfonium as the methyl donor. Forexample, enzymes derived from bacteria, yeasts, rats or the like can beused.

[0094] There is no particular limitation regarding the method forquantitatively determining D-methionine, but it is preferable todetermine it enzymatically with a D-amino acid converting enzyme. Morepreferably, D-amino acid oxidase [EC 1.4.3.3] is used. The inventors ofthe present invention have unexpectedly made it evident thatD-methionine methyl sulfonium, which is a D-amino acid, substantiallycannot serve as a substrate of D-amino acid oxidase. Thus, if theD-amino acid converting enzyme does not react with D-methionine methylsulfonium, or even though this enzyme reacts therewith, if the enzymehas a sufficiently lower reactivity than that with respect toD-methionine, the produced D-methionine can be determined withoutremoving the D-methionine methyl sulfonium that remains after thereaction with homocysteine methyltransferase from the reaction system.In addition to D-amino acid oxidase, D-amino acid acetyltransferase [EC2.3.1.36], D-amino acid dehydrogenase [EC 1.4.99.1], and the like, whichhave the similar properties, can be used (FIG. 2).

[0095] As shown in FIG. 2, when D-methionine is reacted with D-aminoacid oxidase, hydrogen peroxide is produced. This hydrogen peroxide isled to an oxidative color-developing agent commonly used in the presenceof an SH reagent so as to be determined colorimetrically, as describedabove. Furthermore, when D-methionine is reacted with D-amino acidacetyltransferase, the produced coenzyme A is led to hydrogen peroxideby acyl-coenzyme A synthetase [EC 6.2.1.3] and an acyl-coenzyme Aoxidase [EC 1.3.3.6], and this hydrogen peroxide can be quantitativelydetermined in the same manner.

[0096] (2) MTase Method II

[0097] When D-methionine is reacted with D-amino acid oxidase or D-aminoacid dehydrogenase, ammonia and 4-methylthio-2-oxobutyric acid areproduced. Homocysteine can be quantitatively determined by determiningthese products.

[0098] Ammonia can be quantitatively determined by reacting the ammoniawith reduced nicotinamide adenine dinucleotide (NADH), reducednicotinamide adenine dinucleotide phosphate (NADPH) or derivativesthereof (hereinafter, referred to as NAD(P)H in this specification),2-oxoglutaric acid and glutamate dehydrogenase ([EC 1.4.1.2], [EC1.4.1.3], or [EC 1.4.1.4]), and determining a decrease in NAD(P)H bymeasuring the absorbance change at 340 nm. Alternatively, an increase inNAD, or NADP or derivatives thereof (hereinafter, referred to as NAD(P)in this specification) can be determined. Examples of the derivatives ofNAD(P)H include thio NAD(P)H, and 3-acetylpyridine adenine dinucleotide(or 3-acetylpyridine dinucleotide phosphate). For the determination ofammonia, in addition to the glutamate dehydrogenase as described above,any dehydrogenase can be used, as long as it can catalyze a reductiveamination reaction utilizing ammonia with NAD(P)H as a coenzyme. Forexample, leucine dehydrogenase ([EC 1.4.1.9]), alanine dehydrogenase([EC 1.4.1.1]), serine dehydrogenase ([EC 1.4.1.7]), valinedehydrogenase ([EC 1.4.1.8]), and glycine dehydrogenase ([EC 1.4.1.10])can be used. As a cosubstrate of these dehydrogenases, an ammonium salt,a 2-oxo acid or the like can be used. Examples of the 2-oxo acid includepyruvic acid, 2-oxobutyric acid, 2-oxoisocaproic acid, 2-oxoisovalericacid, 2-oxovaleric acid, 2-oxocaproic acid, glyoxylic acid, andhydroxypyruvic acid, in addition to 2-oxoglutaric acid as describedabove.

[0099] Ammonia can be quantitatively determined by utilizing a Nessler'sreagent, a pH indicator, an electrode method or other methods.

[0100] 4-Methylthio-2-oxobutyric acid can be quantitatively determinedby reacting the 4-methylthio-2-oxobutyric acid with NADH, ammonia andleucine dehydrogenase [EC 1.4.1.9], and determining, for example, adecrease in NADH by measuring the absorbance change at 340 nm as in thecase of ammonia. It is known that 4-methylthio-2-oxobutyric acid servesas a substrate of leucine dehydrogenase (G. Livesey et al. Methods inEnzymology, 166, 282-288, 1988). For the determination of4-methylthio-2-oxobutyric acid, in addition to the leucine dehydrogenaseas described above, any dehydrogenase can be used, as long as it canreduce 4-methylthio-2-oxobutyric acid. For example, lactatedehydrogenase ([EC 1.1.1.27]) can be used.

[0101] It is known that the method for leading 4-methylthio-2-oxobutyricacid or ammonia to a system employed for quantitative determination bythe absorbance change at 340 nm of NAD(P)H is unlikely to be affected bya reducing agent. Therefore, there is no need of using an SH reagent(Ikeda et al. Rinshokensa (Clinical test), 41, 989-993, 1997). Anexample of leading them to an NAD(P)H quantitative determination systemin the presence of a reducing agent is a determination method ofcreatine kinase in blood (Rinshokagaku(Clinical Chemistry), 19, 189-208,1990).

[0102] In the determination system in which ammonia or4-methylthio-2-oxobutyric acid is produced by D-amino acid oxidase, itis possible to remove hydrogen peroxide, which is one of the products,by the use of catalase to avoid the effect thereof.

[0103] The method of the present invention can be employed manually orby automatic analysis. For example, when a conventional automaticanalysis apparatus for a two reagent system is used, the method isdivided into the first process of performing the homocysteinemethyltransferase reaction and the second process of detectingD-methionine, so as to determine homocysteine in a biological sampleeasily.

[0104] In the quantitative determination of homocysteine by the methodof the present invention, the accuracy depends on D-amino acid in thesample, but the amount of D-amino acid in a biological sample is verysmall. However, it has been reported that the amount of D-amino acid canbe increased in a certain kind of disease. Therefore, in order to avoidthe effect of D-amino acids, determination is performed with a reagentprepared in the same manner except that it does not contain homocysteinemethyltransferase, and the result is subtracted from the value obtainedby determination in a sample contained this enzyme so that the amount ofhomocysteine can be determined accurately.

[0105] When the absorbance of NADH is detected, first, a reagent for areductive reaction and the second process is added to a sample to causea reaction, and then a reagent containing homocysteine methyltransferasefor the first process is added thereto to cause a reaction. Then, thedifference in the absorbance at the end of each reaction is obtained,which makes it possible to quantitatively determine homocysteine withoutany effect of endogenous substances such as D-amino acid.

[0106] Moreover, the present invention provides a kit for determininghomocysteine in a sample comprising (a) a thiol compound for reducinghomocysteine, (b) a homocysteine-converting enzyme and a homocysteinecosubstrate for reaction with the reduced homocysteine (first process),and (c) (i) an SH reagent and an oxidative color-developing agent or(ii) dehydrogenase using NAD(P)H as a coenzyme and a cosubstrate or acolor-developing agent in accordance with the properties of thedehydrogenase for determination of the residual homocysteine cosubstrateor the produced homocysteine-converting enzyme product (second process).In order to perform the reduction process and the first process at thesame time, (a) and (b) can be contained together. In particular, when(c) (ii) is used, (a), (b), and (c) can be contained together. As in thecase of (A) described above, for the purpose of avoiding the effect ofreaction intermediates derived from inosine contained in a sample on thedetermined value, purine nucleoside phosphorylase, phosphoric acid,xanthine oxidase, catalase, and, if necessary, uricase can be containedin the reagent for the first process. Furthermore, instead of catalase,or in addition to catalase, peroxidase and one of a Trinder reagent or acoupler reagent can be contained.

EXAMPLE Example 1

[0107] Effect of N-ethylmaleimide on adenosine deaminase derived frombovine small intestine and inosine determination system enzyme

[0108] The effect on the second process in which N-ethylmaleimide (NEM)was used as the SH reagent was examined.

[0109] First, 100 mM of phosphate buffer (pH7.4), 1 mM of adenosine, 0.4U/mL of purine nucleoside phosphorylase (manufactured by Toyobo Co.,Ltd.), 3 U/mL of xanthine oxidase (manufactured by Toyobo Co., Ltd.), 11U/mL of peroxidase (manufactured by Toyobo Co., Ltd.), 1 mM of4-aminoantipyrine, and 1 mM ofN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) were mixedand incubated at 37° C. for 5 minutes. Then, 0.001 U/mL of adenosinedeaminase derived from small intestine (manufactured by Sigma-AldrichCorp.) were added thereto, and the absorbance change at 540 nm wasdetected by using a spectrophotometer (UV-2200 manufactured by ShimadzuCorporation). The change of absorbance in a linear portion was 0.0676per minute. Then, 1 mM of N-ethylmaleimide was added and detection wasperformed in the same manner. The absorbance change was 0.0656 perminute. Consequently, it became evident that N-ethylmaleimide (NEM)hardly affected the determination system of adenosine deaminase derivedfrom bovine small intestine and inosine.

Example 2

[0110] Effect of N-ethylmaleimide (NEM) on adenosine deaminase derivedfrom bovine pancreas

[0111] The examination was performed in the same manner as in Example 1except that the adenosine deaminase derived from bovine small intestine(Sigma-Aldrich Corp.) was replaced by adenosine deaminase derived frombovine pancreas (Sigma-Aldrich Corp.). The results were that theabsorbance change for one minute before adding N-ethylmaleimide was0.0751, whereas it was 0.0721 after the addition. Consequently, itbecame evident that N-ethylmaleimide (NEM) also hardly affected theadenosine deaminase derived from bovine pancreas.

Example 3

[0112] Interference of color development by a reducing agent in ahomocysteine determination system by the use of an oxidativecolor-developing agent and the prevention thereof by an SH reagent

[0113] Determination was performed by using a Hitachi 7170 automaticanalysis apparatus (manufactured by Hitachi, Ltd.) at a reactiontemperature of 37° C., a dominant wavelength of 546 nm, and a secondarywavelength of 700 nm. First, 180 μL of a reagent 1 containing 100 mM ofphosphate buffer (pH7.4), 0.009 mM of adenosine, 0.8 U/mL ofS-adenosyl-L-homocysteine hydrolase, 1.5 U/mL of uricase, 4.3 U/mL ofxanthine oxidase, 6.4 U/mL of peroxidase, 2.9 mM of 4-aminoantipyrine,2.3 mM of dithiothreitol and 0.2% Triton X-100 were added to 20 μL of100 mM of phosphate buffer (pH7.4) and allowed to react for about 5minutes. Then, 180 μL of a reagent 2 containing 100 mM of phosphatebuffer (pH7.4), 0.3 U/InL of adenosine deaminase, 1.3 U/mL of purinenucleoside phosphorylase, 1.8 mM of TOOS, 17 mM of N-ethylmaleimide and0.1% Triton X-100 were added thereto and allowed to react further forabout 5 minutes. In parallel to this, the same operation was performedexcept that N-ethylmaleimide was not contained in the reagent 2, and thetwo reaction processes were compared. The results are shown in FIG. 3.

[0114] As seen from FIG. 3, in the absense of N-ethylmaleimide (NEM),the color development was significantly interfered, and determinationwas not possible at all (◯). On the other hand, in the presense ofN-ethylmaleimide (NEM), which is an SH reagent, a color was developedwithout being affected by the reducing reagent ().

Example 4

[0115] Dose dependence in the case of using a homocystine specimen as asample

[0116] Determination was performed by using the Hitachi 7170 automaticanalysis apparatus at a reaction temperature of 37° C., a dominantwavelength of 546 nm, and a secondary wavelength of 700 nm. First, 180μL of a reagent 1 containing 100 mM of phosphate buffer (pH7.4), 0.7U/mL of purine nucleoside phosphorylase, 2.7 U/mL of xanthine oxidase,10 U/mL of peroxidase, 0.009 mM of adenosine, 1.8 mM of4-aminoantipyrine, 1.8 mM of dithiothreitol, 0.4 U/mL ofS-adenosyl-L-homocysteine hydrolase, and 0.5% Triton X-100 were added to20 μL of a sample containing 100 mM of phosphate buffer (pH7.4) and 0,15.625, 31.25, 62.5 or 125 μM of homocystine (0 to 250 μM in terms ofhomocysteine) and allowed to react for about 5 minutes. Then, 180 μL ofa reagent 2 containing 100 mM of phosphate buffer (pH7.4), 2 mM of TOOS,6 mM of N-ethylmaleimide, and 0.2 U/mL of adenosine deaminase were addedthereto and allowed to react further for about 5 minutes. The absorbancechange was obtained from the detection point 16 to 34 of the Hitachi7170. The results are shown in FIG. 4. As seen from FIG. 4, there is alinear relationship between the absorbance change and the homocysteineconcentration up to 125 μM, and it is found that the determination ofhomocysteine is possible.

Example 5

[0117] Homocysteine determination in a serum sample (SAHase method)

[0118] Determination was performed by using the Hitachi 7170 automaticanalysis apparatus with a three reagent system at a reaction temperatureof 37° C., a dominant wavelength of 546 nm, and a secondary wavelengthof 700 nm. First, 50 μL of a reagent 1 containing 100 mM of phosphatebuffer (pH7.4), 7 mM of dithiothreitol, 0.028 mM of adenosine and 0.3%Triton X-100 were added to 20 μL of a sample containing normal controlserum SERACLEAR HE added with 0, 2.5, 5, 10, 20, 30, 40 or 50 μM ofhomocystine (0 to 100 μM in terms of homocysteine). Then, about 80seconds later, 130 μL of a reagent 2 containing 100 mM of phosphatebuffer (pH7.4), 2 U/mL of uricase, 1.6 U/mL of purine nucleosidephosphorylase, 5.9 U/mL of xanthine oxidase, 22 U/mL of peroxidase, 4 mMof 4-aminoantipyrine, 0.5 mM of dithiothreitol, 1.1 U/mL ofS-adenosyl-L-homocysteine hydrolase, and 0.1% Triton X-100 were addedthereto and allowed to react for about 8 minutes, so that adenosylhomocysteine was produced. At the same time, the effect of reactionintermediates derived from inosine contained in the sample on thedetermined value was avoided. Furthermore, 180 μL of a reagent 3containing 100 mM of phosphate buffer (pH7.4), 1.8 mM of TOOS, 17 mM ofN-ethylmaleimide, 0.23 U/mL of adenosine deaminase, and 0.1% TritonX-100 were added thereto and allowed to react further for about 5minutes. The absorbance was detected immediately before the addition ofthe reagent 3 (at detection point 33 of Hitachi 7170) and 5 minutesafter the addition (at detection point 50 of Hitachi 7170) and thechange amount was obtained.

[0119] At the same time, a sample blank was determined. The sameoperation was performed, except that the reagent 2 (hereinafter,referred to as an “SAHase-added reagent”) containingS-adenosyl-L-homocysteine hydrolase (which may be referred to as“SAHase” in the following) was replaced by a reagent having the samecomponents as those of reagent 2 except SAHase (hereinafter, referred toas a “reagent without SAHase”). As described above, since a slightamount of adenosine deaminase is contaminated in the SAHase specimen, itis necessary to eliminate the effect. Therefore, at the same time, using100 mM phosphate buffer (pH7.4) as a sample, a blank of the SAHase-addedreagent and a blank of the reagent without SAHase were detected and theratio thereof was used as a correction coefficient with respect to theeffect of the contaminated adenosine deaminase.

[0120]FIG. 5 shows the result of plotting (A) the observed value usingthe reagent without SAHase, (B) the value corrected by multiplying theobserved value using the SAHase-added reagent by the correctioncoefficient (hereinafter, the corrected value of the SAHase-addedreagent), and (A-B) the value obtained by subtracting the correctedvalue of the SAHase-added reagent from the observed value of the reagentwithout SAHase, against the amount of the homocysteine added. Theobserved value of the reagent without SAHase is constant, regardless ofthe amount of the homocysteine added (A:◯), whereas the corrected valueof the SAHase-added reagent decreases depending on the amount ofhomocysteine added (B:). Therefore, the value obtained by subtractingthe corrected value of the SAHase-added reagent from the observed valueof the reagent without SAHase depends on the amount of the homocysteineadded (A-B:▴). On the other hand, a phosphate buffer containing 31.25 μMhomocystine (62.5 μM in terms of homocysteine) was used as a sample, andeach of the SAHase-added reagent and the reagent without SAHase is usedas the reagent 2 for determination in the same manner and the valueswere corrected, so that the value (the difference in the absorbance)obtained by subtracting the corrected value of the SAHase-added reagentfrom the observed value of the reagent without SAHase was obtained, anda factor indicating the amount of the homocysteine per the absorbancedifference was calculated. The amount of the homocysteine in each samplewas calculated by multiplying the value (A-B in FIG. 5:▴) obtained bysubtracting the corrected value of the SAHase-added reagent from theobserved value of the reagent without SAHase in the serum-based sampleby the factor. FIG. 6 shows the results of plotting the amount of thehomocysteine against the amount of the homocysteine added ().

[0121] As seen from FIG. 6, it is possible to determine the totalhomocysteine in serum until about 80 μM in the serum-based sample whileavoiding the influence of the reaction intermediates derived frominosine contained in the sample and the endogenous adenosine or the likeon the observed value.

Example 6

[0122] Effect of cysteine and methionine on the homocysteinedetermination system (SAHase method)

[0123] Determination was performed in the same manner as in Example 5except that a sample containing normal control serum SERACLEAR HE and 27μM of homocystine (54 EM in terms of homocysteine), and samples in which500 μM of methionine or 1000 μM of cysteine was added to this samplewere used. As shown in FIG. 7, homocysteine can be determined accuratelyup to 1000 μM without any interference by cysteine and methionine.

Example 7

[0124] Correlation between the the Present Invention (SAHase method) andthe Conventional HPLC Method

[0125] For 53 serum samples, homocysteine was determined by the SAHasemethod. The determination was performed in the same manner as in Example5, except that the followings were used: a control serum SERACLEAR HEcontaining 52.3 μM homocysteine as the standard; DA-67 as thecolor-developing reagent; and a dominant wavelength of 660 nm and asecondary wavelength of 750 nm. In other words, first, 50 μL of thereagent 1 containing 100 mM of phosphate buffer (pH 7.4), 7 mM of DTT,0.015 mM of adenosine and 0.05% Triton X-100 were added to 10 μL of aserum sample. Then, about 80 seconds later, 130 μL of a reagent 2containing 100 mM of phosphate buffer (pH7.4), 2 U/mL of uricase, 1.6U/mL of purine nucleoside phosphorylase, 6 U/mL of xanthine oxidase, 150U/mL of catalase, 0.84 mM of DA-67, 0.5 mM of DTT, 1.1 U/mL of SAHase,and 0.05% Triton X-100 were added thereto and allowed to react for about8 minutes. Furthermore, 180 μL of a reagent 3 containing 100 mM ofphosphate buffer (pH7.4), 11 U/mL of peroxidase, 17 mM ofN-ethylmaleimide, 0.23 U/mL of adenosine deaminase, and 0.05% TritonX-100 were added thereto and allowed to react further for about 5minutes. The detection point and the calibration were the same as inExample 5.

[0126] On the other hand, the homocysteine in the same samples wasdetermined by the HPLC method (consigned to SRL Inc.).

[0127]FIG. 8 shows the results of plotting the value obtained by theHPLC method on the horizontal axis and the value obtained by the methodof the present invention (SAHase method) on the vertical axis. As seenfrom FIG. 8, a very good correlation is obtained, and the homocysteinein the sample can be determined accurately by the method of the presentinvention.

Example 8

[0128] Preparation of homocysteine methyltransferase

[0129] The method of S. K. Shapiro (Methods Enzymol., 17 Pt.B, 400-405,1971) was partially modified to prepare a homocysteine methyltransferaseenzyme solution in the following manner.

[0130] First, 250 g of baker's yeast (manufactured by Oriental YeastCo., Ltd.) was suspended in 125 ml of distilled water, and heated to 37°C., and then 16.3 g of sodium hydrogencarbonate and 44 ml of toluenewere added thereto with stirring. The mixture was incubated for 90minutes with stirring at 37° C., and then an equal volume of ice-cooleddistilled water was added thereto, and the mixture was cooled rapidly.This solution was centrifuged at 7000 rpm for 30 minutes. Thesupernatant was filtered through a paper towel and the filtrate wasfurther centrifuged at 9000 rpm for 90 minutes, and the supernatant wascollected.

[0131] Then, L-methionine was added with stirring under ice cooling suchthat the final concentration was 0.5%, and was dissolved over about 60minutes. The temperature of the solution was kept at 3° C. or less, andethanol cooled to about −20° C. was added at a rate of 20 mL/min withstirring. At the point when the concentration of ethanol reached about25%, cooling was started by lowering the temperature of the cooling bathto −10° C. or less by using salt-ice. Then, ethanol was added in thesame manner until the final concentration reached 53%, and then thesolution was allowed to stand at −20° C. for 16 hours. Then, thesolution was centrifuged at 9000 rpm and −10° C. for 60 minutes. Thesupernatant was collected, and ethanol was added under the sameconditions until the final concentration reached 70% while cooling to−10° C. or less by using salt-ice. After the addition, the solution wasallowed to stand for one hour, and centrifuged at 9000 rpm and −10° C.for 90 minutes.

[0132] The resultant precipitate was dissolved in about 7 mL of 10 mM ofpotassium phosphate buffer (pH 6.8). The dissolved solution was dialyzedtwice against the same buffer, and then concentrated to about 1.5 mLwith a centrifugal concentrator (Centriprep-10 manufactured by Amicon).Thus, an enzyme solution was obtained.

[0133] Then, the homocysteine methyltransferase activity of, theobtained enzyme solution was determined in the following manner. First,60 mM of phosphate buffer (pH 7.4), 10% of the enzyme solution, 1 mM ofdithiothreitol, 0.2 mM of homocystine (H-6010 manufactured bySigma-Aldrich Corp.), and 0.4 mM of iodinated L-methionine methylsulfonium (27794-0250 manufactured by Across) or brominated D-methioninemethyl sulfonium (29939-0010 manufactured by Across) were mixed andallowed to react at 37° C. for 2 hours. The mixture was spotted on athin layer plate (plate for thin layer chromatography) in an amount ofabout 8 μL for each spot, and was developed with 95% ethanol-28% aqueousammonia (77:23, v/v), and then color development was performed byninhydrin. As a result, it was confirmed that in either case whereL-methionine methyl sulfonium or D-methionine methyl sulfonium was usedas the substrate, methionine was contained in the reaction product. Onthe other hand, it was confirmed that when homocysteine, which is amethyl acceptor, was removed from the reaction system, methionine wasnot produced.

[0134] From the above, it was confirmed that the obtained enzymesolution has homocysteine methyltransferase activity, and that thisenzyme solution can utilize not only L-methionine methyl sulfonium, butalso D-methionine methyl sulfonium as the methyl donor.

Example 9

[0135] Reactivity of D-amino acid oxidase derived from porcine kidneywith respect to D-methionine and D-methionine methyl sulfonium

[0136] The reactivity of D-amino acid oxidase derived from porcinekidney with respect to D-methionine and D-methionine methyl sulfoniumwas determined by using a Hitachi 7170 automatic analysis apparatus inthe following manner. First, 200 μL of a reagent 1 containing 92 mM ofphosphate buffer (pH7.4), 1.3 mM of 4-aminoantipyrine, and 3.3 U/mL ofperoxidase were added to 10 μL of a sample containing 1.3 mM ofD-methionine and allowed to react at 37° C. for about 5 minutes. Then,50 μL of a reagent 2 containing 69 mM of phosphate buffer (pH7.4), 5.2mM of TOOS, 2.6 U/mL of D-amino acid oxidase derived from porcine kidney(manufactured by Sigma-Aldrich Corp.), and 2.6 mM of flavin adeninedinucleotide (FAD) were added thereto and allowed to react further forabout 5 minutes at 37° C. The absorbance change was detected at adominant wavelength of 546 nm and a secondary wavelength of 700 nm.Furthermore, a sample containing 1.3 mM of brominated D-methioninemethyl sulfonium and a sample containing 1.3 mM of D-methionine and 1.3mM of brominated D-methionine methyl sulfonium were determined exactlyin the same manner. FIG. 9 shows the time course of the reaction. TheD-amino acid oxidase derived from porcine kidney was hardly reacted withD-methionine methyl sulfonium (Δ), compared with D-methionine (). Itwas also found that the reactivity with D-methionine was not changedeven in the presence of D-methionine methyl sulfonium (▴).

Example 10

[0137] Reactivity of D-amino acid oxidase derived from fungi withrespect to D-methionine and D-methionine methyl sulfonium

[0138] The reactivity was determined in the same manner as in Example 9,except that D-amino acid oxidase derived from porcine kidney wasreplaced by D-amino acid oxidase derived from fungi (Fusarium,manufactured by Ikedatohka Kogyo). FIG. 10 shows the time course of thereaction. It was evident that the D-amino acid oxidase derived fromfungi also was hardly reacted with D-methionine methyl sulfonium (Δ),compared with D-methionine (). Moreover, it was also found that thereactivity with D-methionine was not changed even in the presence ofD-methionine methyl sulfonium (▴).

Example 11

[0139] Effect of N-ethylmaleimide on a D-methionine determination systememploying an oxidative color-developing agent in the presence of areducing agent

[0140] Next, D-methionine was determined by using a Hitachi 7170automatic analysis apparatus in the following manner. First, 200 μL of areagent 1 containing 100 mM of phosphate buffer (pH7.4), 1 mM of TOOS,and 0.05% Triton X-100 were added to 20 μL of 0, 0.0625, 0.125, 0.25,0.5 or 1 mM of D-methionine containing 5 mM of dithiothreitol (DTT) andallowed to react at 37° C. for about 5 minutes. Then, 50 μL of a reagent2 containing 100 mM of phosphate buffer (pH7.4), 4 mM of4-aminoantipyrine, 1 U/mL of D-amino acid oxidase, 17.6 U/mL ofperoxidase, 1 mM of FAD and 0.05% Triton X-100 were added thereto andallowed to react further for about 5 minutes at 37° C. The absorbancechange (at a dominant wavelength of 546 nm and a secondary wavelength of700 nm) from the detection point 16 to 34 was measured. Then, a reagentwas prepared in the same manner except that 2.8 mM of N-ethylmaleimide(NEM) was added to the reagent 1, and the determination was performedfor the case of the NEM addition in the same manner.

[0141] As shown in FIG. 11, when the determination was performed by theuse of the reagent without NEM (◯), D-methionine was not detected at allbecause of the dithiothreitol contained in the sample. On the otherhand, when the determination was performed by the use of the reagentwith NEM (), a linear dose dependence was recognized and it was foundthat the D-methionine can be determined.

Example 12

[0142] Examination of the dose dependence by the use of a homocysteinespecimen in the method of the present invention (MTase method I)

[0143] First, 0, 100, or 200 μM of homocystine (0, 200 or 400 μM interms of homocysteine, respectively), 86 mM of phosphate buffer (pH7.4), 10% of the enzyme solution, 4 mM of dithiothreitol and 1.5 mM ofbrominated D-methionine methyl sulfonium were reacted at 37° C. for 90minutes.

[0144] The amount of the D-methionine in the reaction mixture wasdetermined by using a Hitachi 7170 automatic analysis apparatus in thefollowing manner. First, 200 μL of a reagent 1 containing 100 mM ofphosphate buffer (pH7.4), 1 mM of TOOS, and 1.7 mM of N-ethylmaleimide(NEM) were added to 30 μL of the sample (reaction mixture) and allowedto react at 37° C. for about 5 minutes. Then, 50 μL of a reagent 2containing 100 mM of phosphate buffer (pH7.4), 4 mM of4-aminoantipyrine, 1 U/mL of D-amino acid oxidase, 17.6 U/mL ofperoxidase, and 0.2 mM of FAD were added thereto and allowed to reactfurther for about 5 minutes at 37° C. The absorbance change (at adominant wavelength of 546 nm and a secondary wavelength of 700 nm) fromthe detection point 16 to 34 was measured. The results are shown in FIG.12 with the homocystine concentration on the horizontal axis and theabsorbance change (×10000) on the vertical axis.

[0145] As seen from FIG. 12, the absorbance change increases dependingon the homocystine dose (). On the other hand, when D-methionine methylsulfonium was not added (Δ), and when the enzyme was not added (□), thedose dependence was not seen.

Example 13

[0146] Examination of the dose dependence of serum homocysteine in themethod of the present invention (MTase method I)

[0147] First, 50 μL of a treatment solution containing 50 mM ofphosphate buffer (pH 7.4), 30% of the enzyme solution, 15 mM ofdithiothreitol and 3 mM of brominated D-methionine methyl sulfonium wereadded to 100 μL of a sample in which 0, 10, 20, 30, 40 or 50 μM ofhomocystine (0 to 100 μM in terms of homocysteine) was added to normalcontrol serum SERACLEAR HE (AZWELL Inc.), mixed therewith, and allowedto react at 37° C. for 90 minutes.

[0148] The amount of the D-methionine in the reaction mixture wasdetermined by using a Hitachi 7170 automatic analysis apparatus in thefollowing manner. First, 200 μL of a reagent 1 containing 100 mM ofphosphate buffer (pH7.4), 1 mM of TOOS, 2.8 mM of NEM and 0.05% TritonX-100 were added to 20 μL of the sample (reaction mixture) and allowedto react at 37° C. for about 5 minutes. Then, 50 μL of a reagent 2containing 100 mM of phosphate buffer (pH7.4), 4 mM of4-aminoantipyrine, 1 U/mL of D-amino acid oxidase, 17.6 U/mL ofperoxidase, 1 mM of FAD and 0.05% Triton X-100 were added thereto andallowed to react further for about 5 minutes at 37° C. The absorbancechange (at a dominant wavelength of 546 nm and a secondary wavelength of700 nm) from the detection point 16 to 34 was measured. The results areshown in FIG. 13 with the added homocystine concentration on thehorizontal axis and differences in absorbance on the vertical axis, inwhich the differences were obtained by subtracting the absorbance change(×10000) in the sample without homocystine from the absorbance change(×10000) in each sample.

[0149] As seen from FIG. 13, the dose dependence on the amount of thehomocystine added was also found in the serum sample (). On the otherhand, when D-methionine methyl sulfonium was not added (◯), such adependence was not found. The above-described results make it evidentthat the amount of the homocysteine in the sample can be quantitativelydetermined.

Example 14

[0150] Determination by the use of a Highly Sensitive Color-developingAgent

[0151] Determination was performed exactly in the same manner as inExample 13, except that a TOOS and 4-aminoantipyrine system as thecolor-developing agent for quantitative determination of D-methioninewere replaced by TPM-PS, which is a highly sensitive color-developingagent. More specifically, a reagent obtained by removing TOOS from thereagent 1 for quantitative determination of D-methionine, and a reagentobtained by removing 4-animo antipyrine from the reagent 2 and adding 2mM of TMP-PS instead were used for determination. As shown in FIG. 14,the determination by the use of TMP-PS can be performed with highersensitivity than the determination by the use of the TOOS and4-aminoantipyrine system.

Example 15

[0152] Correlation between the Present Invention (MTase Method I) andthe Conventional HPLC Method.

[0153] For 35 serum samples, homocysteine was determined by the MTasemethod I. A saline was used as a reagent blank, and a saline containing50 μM homocysteine was used as a standard. First, 50 μL of 35 mM ofphosphate buffer (pH 7.0) containing 9.6 U/L homocysteinemethyltransferase, 15 mM DTT, 1.5 mM of brominated D-methionine methylsulfonium and 0.5 mM of zinc bromide were added to 100 μL of a sampleand allowed to react at 37° C. for 90 minutes. It should be noted that 1U of homocysteine methyltransferase is the amount of enzyme thatcatalyzes D-methionine synthesis in an amount of 1 umol per minute whenD-methionine methyl sulfonium is used as the methyl donor andhomocysteine is used as a methyl acceptor. The same sample was reactedwith a reagent that does not contain homocysteine methyltransferase inthe same manner. Then, 150 μL of a solution containing 18 mM of NEM wasadded thereto to stop the reaction. The amount of the D-methionine inthe reaction mixture was determined by using a Hitachi 7170 automaticanalysis apparatus in the following manner. First, 150 μL of 96 mM ofphosphate buffer (pH7.0) containing 0.48 mM of TOOS were added to 30 μLof the reaction mixture and allowed to react at 37° C. for about 5minutes. Then, 100 μL of 90 mM of phosphate buffer (pH7.0) containing0.7 mM of 4-aminoantipyrine, 1.4 U/mL of D-amino acid oxidase, 4.4 U/mLof peroxidase, and 1 mM of FAD were added thereto and allowed to reactfurther for about 5 minutes at 37° C. The absorbance change (at adominant wavelength of 546 nm and a secondary wavelength of 700 nm) fromthe detection point 16 to 34 was measured. The amount of thehomocysteine in the sample was calculated using a value obtained bysubtracting the absorbance change in the reagent without homocysteinemethyltransferase (sample blank) from the absorbance change in thereagent with this enzyme.

[0154] On the other hand, the homocysteine in the same sample wasdetermined by the HPLC method (consigned to SRL Inc.).

[0155] The results are shown in FIG. 15 with the value obtained by theHPLC method on the horizontal axis and the value obtained by the methodof the present invention (MTase method I) on the vertical axis. As seenfrom FIG. 15, a very satisfactory correlation is obtained, and thehomocystine in the samples can be determined accurately by the method ofthe present invention.

Example 16

[0156] Dose dependency in the D-methionine determination system usingD-amino acid oxidase and leucine dehydrogenase and the effect of areducing agent, DTT

[0157] The D-methionine was determined by the use of a Hitachi 7170automatic analysis apparatus in the following manner. First, 180 μL of areagent 1 containing 50 mM of TAPS(N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid; manufacturedby Dojin Kagaku Kenkyusho) (pH8.5), 990 mM of ammonium chloride(manufactured by Nakarai), 3.4 U/mL leucine dehydrogenase (manufacturedby Wako Pure Chemical Industries, Ltd.), 32 U/ml of catalase(manufactured by Roche) and 0.16 mM of NADH (manufactured by OrientalYeast Co., Ltd.) were added to 30 μL of a sample containing 0, 25, 50,100, 200 or 400 μM of D-methionine in a saline (0.9% NaCl) and allowedto react at 37° C. for about 5 minutes. Then, 40 μL of a reagent 2containing 50 mM of TAPS (pH8.5), 990 mM of ammonium chloride, 5 U/mL ofD-amino acid oxidase (derived from porcine kidney, manufactured byKikkoman) and 0.1 mg/mL of FAD were added thereto and allowed to reactfurther for about 5 minutes at 37° C. The absorbance change (at adominant wavelength of 340 nm and a secondary wavelength of 405 nm) ofNADH from the detection point 16 to 34 was measured. Next, a reagent wasprepared exactly in the same manner except that 10 mM of DTT was addedto the reagent 1, and the determination by the use of DTT was performedin the same manner.

[0158] The results are shown in FIG. 16 with the D-methionineconcentration on the horizontal axis and the absorbance change at 340 nm(secondary wavelength of 405 nm) on the vertical axis.

[0159] As seen from FIG. 16, when DTT was not added (Δ), theD-methionine dose-dependent linear absorbance change was observed inthis determination system, so that D-methionine can be quantitativelydetermined. When DTT was added (◯), the determination of D-methioninewas not affected by DTT.

Example 17

[0160] Dose dependency in the homocysteine determination system (MTasemethod II) using homocysteine methyltransferase, D-amino acid oxidaseand leucine dehydrogenase

[0161] Homocysteine was determined by the use of a Hitachi 7170automatic analysis apparatus in the following manner. First, 180 μL of areagent 1 containing 50 mM of TAPS (pH8.5), 990 mM of ammonium chloride,10 mM of DTT, 0.5 U/mL of homocysteine methyltransferase (derived fromyeast, obtained from Ozeki), 0.6 mM of brominated D-methionine methylsulfonium, 5 U/mL leucine dehydrogenase (manufactured by Wako PureChemical Industries, Ltd.), 32 U/ml of catalase and 0.16 mM of NADH wereadded to 30 μL of a sample containing 0, 12.5, 25, 50, or 100 μM ofhomocystine (0, 25, 50, 100 or 200 μM in terms of homocysteine) in asaline (0.9% NaCl) and allowed to react at 37° C. for about 5 minutes.Then, 40 μL of a reagent 2 containing 50 mM of TAPS (pH8.5), 990 mM ofammonium chloride, 7.5 U/mL of D-amino acid oxidase (manufactured byKikkoman) and 0.1 mg/mL of FAD were added thereto and allowed to reactfurther for about 5 minutes at 37° C. The absorbance change (at adominant wavelength of 340 nm and a secondary wavelength of 405 nm) inNADH from the detection point 16 to 34 was measured. Next, determinationwas performed exactly in the same manner as above except that a samplein which 0, 12.5, 25, 50 or 100 μM of homocystine was added to a normalcontrol serum SERACLEAR HE was used as the sample.

[0162] The results are shown in FIG. 17 with the concentration of theadded homocysteine on the horizontal axis and the absorbance change at340 nm (secondary wavelength of 405 nm) on the vertical axis.

[0163] As seen from FIG. 17, in both the cases of the homocysteinespecimen () and the serum sample (▴), it can be seen that theabsorbance change depends on the dose of the homocysteine added, so thatit is evident that also in this determination system, homocysteine canbe quantitatively determined.

Example 18

[0164] Dose dependency in the homocysteine determination system (MTasemethod II) using homocysteine methyltransferase, D-amino acid oxidaseand glutamate dehydrogenase

[0165] Homocysteine was determined by using a Hitachi 7170 automaticanalysis apparatus in the following manner. First, 180 μL of a reagent 1containing 100 mM of Tris buffer (pH8.0), 10 mM of DTT, 0.6 mM ofbrominated D-methionine methyl sulfonium, 2 U/mL D-amino acid oxidase(manufactured by Kikkoman), 0.03 mg/mL of FAD, 32 U/ml of catalase, 8U/mL of glutamate dehydrogenase (manufactured by Toyobo Co., Ltd.), 8 mMof 2-oxoglutaric acid (manufactured by Nakarai), and 0.16 mM of NADHwere added to 30 μL of a sample containing 0, 12.5, 25, 50, or 100 μM ofhomocystine (0, 25, 50, 100 or 200 μM in terms of homocysteine) in asaline (0.9% NaCl) and allowed to react at 37° C. for about 5 minutes.Then, 40 μL of a reagent 2 containing 100 mM of Tris buffer (pH8.0) and2.1 U/mL of homocysteine methyltransferase (derived from yeast, obtainedfrom Ozeki) were added thereto and allowed to react further for about 5minutes at 37° C. The absorbance change (at a dominant wavelength of 340nm and a secondary wavelength of 405 nm) in NADH from the detectionpoint 16 to 34 was measured. Next, determination was performed in thesame manner as above except that a sample in which 0, 12.5, 25, 50 or100 μM of homocystine was added to a normal control serum SERACLEAR HEwas used.

[0166] The results are shown in FIG. 18 with the concentration of theadded homocysteine on the horizontal axis and the absorbance change at340 nm (secondary wavelength of 405 nm) on the vertical axis.

[0167] As seen from FIG. 18, in both the cases of the homocysteinespecimen () and the serum sample (▴), it can be seen that theabsorbance change depends on the dose of the homocysteine added. It isevident that also in this determination system, homocysteine can bequantitatively determined.

Example 19

[0168] Quantitativity of 4-methylthio-2-oxobutyric acid by lactatedehydrogenase

[0169] 4-Methylthio-2-oxobutyric acid was determined by using a Hitachi7170 automatic analysis apparatus in the following manner. First, 180 μLof a reagent 1 containing 200 mM of phosphate buffer (pH7.0) and 0.16 mMof NADH were added to 30 μL of a sample containing 0, 50, 100, 200 or400 μM of 4-methylthio-2-oxobutyric acid in a saline (0.9% NaCl) andallowed to react at 37° C. for about 5 minutes. Then, 40 μL of a reagent2 containing 200 mM of phosphate buffer (pH7.0) and 65 U/mL of lactatedehydrogenase (derived from porcine heart, manufactured by OrientalYeast) were added thereto and allowed to react further for about 5minutes at 37° C. The absorbance change (at a dominant wavelength of 340nm and a secondary wavelength of 405 nm) in NADH from the detectionpoint 16 to 34 was measured.

[0170] The results are shown in FIG. 19 with the4-methylthio-2-oxobutyric acid concentration on the horizontal axis andthe absorbance change at 340 nm (secondary wavelength of 405 nm) on thevertical axis.

[0171] As seen from FIG. 19, it can be seen that the absorbance changedepends on the dose of the 4-methylthio-2-oxobutyric acid, so that it isevident that the lactate dehydrogenase uses 4-methylthio-2-oxobutyricacid as the substrate, and also in this determination system where thelactate dehydrogenase is used instead of the leucine dehydrogenase andthe glutamate dehydrogenase in Examples 17 and 18, homocysteine can bequantitatively determined.

Industrial Applicability

[0172] The present invention allows homocysteine in a biological sample,in particular, in body fluids such as blood and urine to be detected andquantitatively determined rapidly and simply and with high sensitivity.

1. A method for detecting or determining homocysteine in a sample,comprising: (a) reducing the homocysteine in the sample by a thiolcompound, (b) reacting the reduced homocysteine with ahomocysteine-converting enzyme and a homocysteine cosubstrate, therebyproducing a homocysteine converting enzyme product, and (c) detecting ordetermining the residual homocysteine cosubstrate, the producedhomocysteine-converting enzyme product or an enzyme reaction productthereof by (i) oxidizing the residual homocysteine cosubstrate, theproduced homocysteine-converting enzyme product or an enzyme reactionproduct thereof in the presence of an SH reagent to produce hydrogenperoxide and detecting or determining the produced hydrogen peroxide bycolor development using an oxidative color-developing agent or (ii)reacting the residual homocysteine cosubstrate, the producedhomocysteine-converting enzyme product or an enzyme reaction productthereof with a D-amino acid converting enzyme to produce an oxo acidand/or ammonia and detecting or determining the produced oxo acid and/orammonia.
 2. The method of claim 1, wherein the homocysteine-convertingenzyme in the step (b) is S-adenosyl-L-homocysteine hydrolase, and thehomocysteine cosubstrate in the steps (b) and (c) is adenosine.
 3. Themethod of claim 2, wherein the step (c) of detecting or determining theadenosine is a step of detecting or determining the adenosine byreacting the adenosine with adenosine deaminase, phosphoric acid, purinenucleoside phosphorylase, and xanthine oxidase to produce hydrogenperoxide and detecting or determining the produced hydrogen peroxide bycolor development using peroxidase and an oxidative color-developingagent.
 4. The method of claim 1, wherein the homocysteine-convertingenzyme in the step (b) is a methyltransferase using homocysteine as amethyl acceptor, and the homocysteine cosubstrate in the steps (b) and(c) is a methyl donor.
 5. The method of claim 4, wherein themethyltransferase is homocysteine methyltransferase, and the methyldonor is D-methionine methyl sulfonium.
 6. The method of claim 4 or 5,wherein the D-amino acid converting enzyme is D-amino acid oxidase. 7.The method of any one of claims 4 to 6, wherein in the step (c), theproduced hydrogen peroxide is detected or determined by colordevelopment using peroxidase and an oxidative color-developing agent. 8.The method of any one of claims 4 to 6, wherein in the step (c), adecrease in NAD(P)H or an increase in NAD(P) is detected or determinedby reacting the produced oxo acid and/or ammonia with dehydrogenaseusing NAD(P)H as a coenzyme.
 9. A reagent kit for homocysteinedetermination comprising a thiol compound, a homocysteine-convertingenzyme, a homocysteine cosubstrate, an SH reagent, and an oxidativecolor-developing agent.
 10. The kit of claim 9, wherein thehomocysteine-converting enzyme is S-adenosyl-L-homocysteine hydrolase,and the homocysteine cosubstrate is adenosine.
 11. The kit of claim 10,further comprising adenosine deaminase, phosphoric acid, purinenucleoside phosphorylase, xanthine oxidase, and peroxidase.
 12. Areagent kit for homocysteine determination comprising a thiol compound,a homocysteine-converting enzyme, a homocysteine cosubstrate, and aD-amino acid converting enzyme.
 13. The kit of claim 12, furthercomprising NAD(P)H, dehydrogenase using NAD(P)H as a coenzyme, and anammonium salt or 2-oxo acid as its cosubstrate.
 14. The kit of claim 9,12 or 13, wherein the homocysteine-converting enzyme is amethyltransferase using homocysteine as a methyl acceptor, and thehomocysteine cosubstrate is a methyl donor.
 15. The kit of claim 14,wherein the methyltransferase is homocysteine methyltransferase, and themethyl donor is D-methionine methyl sulfonium.
 16. The kit of any one ofclaims 12 to 15, wherein the D-amino acid converting enzyme is D-aminoacid oxidase.